Antibodies specific for a haemostatic protein, their use for isolating protein, haemostatic compositions devoid of proteolytic cleavage products of the protein

ABSTRACT

A method for the generation of Ca++ independent antibodies against blood coagulation factors is described wherein an antibody selection strategy based upon small peptides comprising target sequences for limited proteolysis is employed. These antibodies which distinguish between intact and cleaved species of haemostatic protein provide novel tools for the isolation of intact haemostatic proteins.

This application and copending application No. 10/228,882 are bothreissues of patent application Ser. No. 08/797,842, U.S. Pat. No.5,932,706, which is a continuation of our co-pending patent applicationfiled on Feb. 8, 1995, assigned Ser. No. 08/381,891, now abandoned andentitled “Antibodies Specific for a Haemostatic Protein. Their Use forIsolating Intact Protein. Haemostatic Compositions Devoid of ProteolyticCleavage Products of the Protein which is 371 of PCT of PCT/NL93/00174filed Aug. 26, 1993.

FIELD OF THE INVENTION

This invention relates to the preparation of therapeutic compositionsconsisting of purified blood coagulation factors for the treatment ofhaemostatic disorders. Said compositions are obtained by affinitychromatography employing antibodies, more in particular monoclonalantibodies, that distinguish between intact and cleaved molecularspecies. Methods are disclosed to obtain such antibodies, which allowthe isolation of vitamin K-dependent blood coagulation proteins,including Factor IX, Factor VII, Protein C or Protein S, as intactpolypeptides, devoid of cleavage products representing activated ordegraded species.

BACKGROUND OF THE INVENTION

Inherited or acquired deficiencies of proteins of the blood coagulationsystem provide a major cause for the occurrence of haemostaticdisorders. Even the lack or shortage of one single component of thissystem may be sufficient to disturb the delicate balance betweenprocoagulant and anticoagulant pathways in a manner resulting in majorclinical signs of bleeding or thrombosis. One of the most commonbleeding disorders is Haemophilia A, which is due to deficiency ordysfunction of the coagulation Factor VIII. Less frequently occurring,but equally severe bleeding disorders include deficiencies of thehaemostatic proteins Factor IX (Haemophilia B), Factor VII, or Factor X.On the other hand, thrombosis may occur as the result of even partial(heterozygous or acquired) deficiency of Protein C or Protein S, whichare major components of a system that acts as an antagonist of thecoagulation pathway (for reviews on haemostatic disorders see A. L.Bloom and D. P. Thomas (Eds.), Haemostasis and Thrombosis, 2nd edition,Churchill-Livingstone, Edinburgh, 1987, pp 393-436 and 452-464).Replacement therapy is considered as a powerful and effective means torestore the haemostatic balance in vivo. For instance, concentratescontaining Factor IX have proven highly valuable blood products whichare life-saving when used to control bleeding in patients suffering fromFactor IX deficiency.

Commercially available Factor IX concentrates (so-called prothrombincomplex concentrates) usually are prepared with ion exchange resins toseparate Factor IX from the other plasma proteins. This techniquehowever, yields Factor IX preparations that also contain a number ofother, closely related haemostatic proteins. These include Factor VII,Factor X, Factor II, Protein C and Protein S, which all belong to theclass of the vitamin K-dependent proteins. The term “vitaminK-dependent” is referring to the fact that these proteins containglutamic acid residues that are carboxylated during biosynthesis in avitamin K-dependent process. Carboxylation provides these proteins withunique Ca²⁺-binding sites that are obligatory for the biologicalactivity of these proteins within the Ca²⁺-dependent haemostaticprocess. Due to these and other structural similarities (see B. Furieand B. C. Furie, Cell vol 53, 1988, pp 505-518), the vitamin K-dependentproteins are readily co-purified. Thus, most Factor IX concentrates arealso containing other haemostatic proteins such as Factor VII, Protein Cand Protein S, and consequently the same concentrates have been used forthe treatment of deficiencies of those proteins as well. However,treatment of bleeding disorders with compositions containinganticoagulant proteins such as Protein C and Protein S is in fact highlyundesired, as is treatment of thromboembolic disease with compositionscontaining Factor IX, Factor VII or other procoagulant components asmajor contaminants. Therefore, the ideal therapeutic composition tocorrect a deficiency of one specific haemostatic protein should consistof solely that single component in an intact conformation and nothingelse except solvent, and sometimes an inert carrier. As a consequence,the purification strategies needed to achieve the desired degree ofpurity have become increasingly complex.

Along with the introduction of advanced, more complex purificationprotocols a novel problem of undesired proteolysis of the target specieswas encountered. Limited proteolysis is a key mechanism in theregulation of a number of biological systems (see H. Neurath and K. A.Walsh, Proc. Natl. Acad. Sci. USA vol 73, 1976, pp 3825-3832). Typicalexamples of such biological systems include the complement system, thefibrinolytic system, and the blood coagulation system. These biologicalcascade systems involve the sequential conversion of intact, inactiveprecursor proteins into active enzymes or cofactors by proteolysis ofone or more specific peptide bonds. On the other hand, feedbackmechanisms exist to maintain these processes under local control andlead to proteolytic inactivation of the target proteins. Accordingly,the components of the coagulation cascade are present in blood plasma ina precursor form lacking biological activity. With regard to replacementtherapy, the presence of coagulation proteins that are no longer intactis troublesome, since after having been subject to limited proteolysis,such cleaved species may bypass the natural, local control ofhaemostasis in vivo. Although natural mechanisms effectively controlproteolysis under physiological conditions, these can no longer bemaintained when haemostatic proteins are isolated from their naturalsource. As such protease-sensitive sequences are exposed within thetertiairy structure of these proteins, they provide easily accessibletargets for proteolysis in a non-physiological environment lackingnatural control mechanisms. Therefore, it is virtually impossible tocompletely prevent partial proteolysis during purification. Uncontrolledproteolysis of these vulnerable proteins is not limited to purificationfrom a natural source as human plasma or fractions thereof, but mayequally occur when the same proteins are obtained by recombinant DNAtechnology from transformed cell lines in vitro, or from biologicalfluids, including milk, of transgenic animals in vivo.

The presence of cleaved species in therapeutic products is clearly notdesired, because the presence of activated proteins may triggerthrombogenic responses of the haemostatic system, whereas the presenceof inactivated proteins leads to products with suboptimal biologicalactivity that may competitively inhibit the reactions to be corrected.Prothrombin complex concentrates contain activated species of virtuallyall vitamin K-dependent coagulation factors, and this has beenestablished as a causative agent for the occurrence of thromboemboliccomplications since the 1970s is (G. C. White et al., Blood vol 49,1977, pp 159-170; J. M. Lusher, Seminars in Hematology vol 28, suppl 6,1991, pp 3-5). Theoretically, in particular those species thatparticipate in the initiation phase of the coagulation system, andthereby are the most amplified in the cascade mechanism, are to beconsidered as the most potent in disturbing the physiologicalhaemostatic balance. Indeed, in vivo studies employing purifiedactivated coagulation factors have identified activated Factor IX (S.Gitel et al., Proc. Natl. Acad. Sci. USA vol 74, 1977, pp 3028-3032) andactivated Factor VII (K. Mertens et al., Thromb. Haemostasis vol 64,1990, pp 138-144) as thrombogenic even in extremely low dosage. This mayraise particular concern for activated forms that are relativelyresistent to inhibition in vivo. Most activated vitamin K-dependentcoagulation factors are subject to almost instantaneous inhibition bythe abundance of protease inhibitors in blood plasma. However, FactorIXa is only slowly inhibited, whereas Factor VIIa and activated ProteinC have in vivo half-lives up to 2 hours (K. Mertens et al., Thromb.Haemostasis vol 64, 1990, pp 138-144; P. C. Comp, Hematology,McGraw-Hill, New York, N.Y., 1990, pp 1290-1303). Thus, upon infusingvitamin K-dependent haemostatic proteins into patients, it should benoted that even traces of activated forms may remain in the patientscirculation sufficiently long to bypass physiological control.Therefore, Protein C and Factors VII and IX are among the proteins thatshould be prevented from being activated, or should be purifiedemploying a strategy that is selective for their intact zymogens. On theother hand, other cleavages can occur that result in the inactivation ofsaid proteins, thus reducing their therapeutic efficacy. For instance,Factor IX can be inactivated by the enzymes thrombin or elastase, andthe cleavage product has no longer the potential of being converted intoa form having Factor IXa activity (A. Takaki et al., J. Clin. Invest.vol 72, 1983, pp 1706-1715; W. Kisiel et al., Blood vol 66, 1985, pp1302-1308). Similarly, the anticoagulant Protein S is readily cleaved bythrombin into a product that no longer has anticoagulant activity (forreview see M. Hessing, Biochem. J. vol 277, 1991, pp 581-592). It thusappears highly important to avoid the occurrence of cleaved, non-intactspecies to reduce side-effects and to improve efficacy of therapeuticconcentrates of these factors.

To better appreciate the structural differences between the intactzymogen species and their cleaved derivatives, the molecular eventsassociated with limited proteolysis of these proteins are now describedin more detail. Table I presents an overview of target sequences forlimited proteolysis as they occur in a number of vitamin K-dependentproteins involved in the haemostatic system. These include:

(a) Human Factor VII: The intact zymogen is a single-chain glycoproteinof 406 amino acids, which is converted to Factor VIIa by the cleavage ofa single bond between residues 152 and 153 (see Table I). This targetsequence can be cleaved by a number of enzymes, including thrombin andFactors IXa, Xa and XIIa, which results in the activated species whichconsists of two polypeptide chains that are held together by a disulfidebond. This Factor VIIa provides a powerful thrombogenic trigger byactivating a number of target substrates, including Factor X and FactorIX (B. Osterud and S. I. Rapaport, Proc. Natl. Acad. Sci. USA vol 74;1977, pp 5260-5264).

(b) Human Factor IX: The intact zymogen is a single-chain glycoproteinof 415 amino acids, which is converted into Factor IXa by Factor XIa orFactor VIIa in two steps. The first step involves the cleavage betweenresidues 145 and 146, which results in the formation of a two-chaininactive intermediate (called Factor IXα) containing a light chain ofresidues 1-145 and a heavy chain of residues 146-415. In the secondstep, the heavy chain of Factor IXα is further cleaved between residues180 and 181, resulting in a 35-residue (146-180) activation peptide andan active enzyme (Factor IXaβ) with the remaining heavy chain ofresidues 181-415. It should be noted that whereas the latter cleavage isrequired to develop the final Factor IXa activity, the prior cleavage ofthe 145-146 bond seems to be an obligatory step in Factor IX activationby Factor XIa or Factor VIIa (U. Hedner and E. W. Davie, in: R. W.Colman et al. (Eds), Hemostasis and Thrombosis, J. B. Lippincott,Philadelphia, 1987, pp 29-38; M. J. Griffith et al., J. Clin. Invest.vol 75, 1985, pp 4-10). Therefore, Factor IXα is even more vulnerablethan the intact Factor IX zymogen with respect to cleavage at the180-181 position and the concurrent formation of a thrombogenic FactorIXa species. In addition to this proteolytic activation, alsoinactivation may occur as the result of cleavage by thrombin orelastase. This proteolytic inactivation involves cleavage betweenresidues 327 and 328, and between residues 338 and 339, resulting inderivatives that can no longer be converted to species with Factor IXprocoagulant activity (see Table I).

(c) Human Protein C: The intact zymogen is a two-chain glycoprotein of419 amino acids, consisting of a light chain of residues 1-155 and aheavy chain of residues 158-419. The zymogen is activated by a singlecleavage in the heavy chain, between residues 169 and 170. The activatedspecies is a powerful anticoagulant, which inactivates Factors Va andVIIIa in a manner requiring the presence of a vitamin K-dependentcofactor, called Protein S.

(d) Human Protein S: The intact species is a single-chain glycoproteinof 635 amino acid residues. The N-terminal portion of the proteincontains two thrombin-sensitive bonds between residues 49-50 and 70-71.After cleavage of Protein S by thrombin, the N-terminal fragment stillis connected to the molecule via a disulfide bond. However, only ProteinS that is uncleaved within the thrombin-sensitive region is active as acofactor for activated Protein C. Therefore, it is preferred to provideintact, uncleaved Protein S in a composition for effective treatment ofheriditary or acquired Protein S deficiency.

In conclusion, a special need exists for purification methods that allowthe selection of uncleaved, intact species from a source containing theintact proteins as well as proteolytic derivatives generated by cleavageat the target peptide bonds that are summarized in Table I.

TABLE I Major cleavage sites in vitamin K-dependent proteins andsynthetic peptides comprising them¹ peptide no.: Factor IX activationsites: 1 PAVPFPCGRVSVSQTSKLTR¹⁴⁵↓AETVFPDVDYVNSTEAETIL SEQ ID NO 9             Q¹³⁹---------------D¹⁵⁴ 2AETILDNITQSTQSFNFTR¹⁸⁰↓VVGGEDAKPGQFPWQVVLNG SEQ ID NO 10            Q¹⁷³--------------K¹⁸⁸ Factor IX degradation sites: 3SGWGRVFHKGRSALVLQYLR³²⁷↓VPLVDRATCLRSTKFTIYNN SEQ ID NO 11            A³²⁰---------------T³³⁵ 4SALVLQYLRVPLVDRATCLR³³⁸↓STKFTIYNNMFCAGFHEGGR SEQ ID NO 12                T³³⁵-------F³⁴² Factor VII activation site: 5YPCGKIPILEKRNASKPQGR¹⁵²↓IVGGKVCPKGECPWQVLLLV SEQ ID NO 13              S¹⁴⁷-----------V¹⁵⁸ Protein C activation site: 6DTEDQEDQVDPR¹⁶⁹↓LIDGKMTRRGDSPWQVVLLD SEQ ID NO 14  E¹⁶⁰-------------------G¹⁷⁹ Protein S degradation sites: 7VFENDPETDYFYPKYLVCLR⁴⁹↓SFQTGLFTAARQSTNAYPDL SEQ ID NO 15          F⁴⁰-------------------A⁵⁹ 8FQTGLFTAARQSTNAYPDLR⁷⁰↓SCVNAIPDQCSPLPCNEDGY SEQ ID NO 16           S⁶²-----------------Q⁷⁹ ¹ (see E.W. Davie et al., in: R.W.Colman et al. (Eds), Hemostasis and Thrombosis, J.B. Lippincott,Philadelphia, 1987, pp 242-267).

DESCRIPTION OF THE PRIOR ART

The development of monoclonal antibody technology has radically changedthe potential for purifying trace proteins from plasma to nearhomogeneity. Monoclonal antibodies can be prepared and selected in sucha manner that they show complete specificity towards an individualplasma protein. When a monoclonal antibody specific for a plasma proteinis immobilized on an inert matrix, it will absorb that plasma proteinspecifically from a mixture containing that plasma protein. Afterwashing the matrix, the plasma protein may be eluted under fairly mildconditions in pure or virtually pure form. This approach has allowed theisolation of pure Factor IX from human blood plasma by immuno-affinitychromatography (A. H. Goodall et al., Protides of the Biological Fluids(Peeters, Ed.) vol 30; Pergamon Press, Oxford, 1982, pp 403-407; K.Mertens et al., Thromb. Haemostasis vol 50; 1983, p 249; K. Mertens,Ph.D. dissertation, State University of Leiden, 1985, pp 83-98). Similarmethods of immuno-affinity chromatography have subsequently becomeavailable for purification of the other vitamin K-dependent haemostaticproteins.

In some cases, a more restricted specificity has been achieved, in thatantibodies may specifically bind to their target vitamin K-dependentprotein in the presence of Ca²⁺-ions. Under these conditions, the targetproteins are in a biologically active conformation by virtue of theinteraction of Ca²⁺-ions with the carboxylated glutamic acid residuesthat are unique to the vitamin K-dependent proteins. When applied in apurification process, such antibodies allow elution of the targetprotein under mild conditions by the Ca²⁺-binding agent EDTA, as hasbeen described for Factor IX (H. A. Liebman et al., Proc. Natl. Acad.Sci. USA vol 82, 1985, pp 3879-3883; K. J. Smith, Blood vol 72, 1988, pp1269-1277) as well as for Protein C (D. J. Stearns et al., J. Biol.Chem. vol 263, 1988, pp 826-832).

However, in spite of their high selectivity, those procedures have notbeen able to distinguish between the intact and activated or degradedcoagulation factors, and as a consequence cleaved products areco-purified with the desired intact, non-activated target product.Moreover, antibodies that are Ca²⁺-dependent have the disadvantage thatCa²⁺-ions cannot be added to most source materials, including plasma orfractions thereof, without simultaneously triggering the Ca²⁺-dependentcoagulation system that leads to proteolytic cleavage of the vitaminK-dependent target proteins. This represents a major drawback for theapplicability of the final therapeutic product as it has beendemonstrated that even small amounts of activated coagulation factorshave been implicated as causative agents for disseminated intravascularcoagulation and thromboembolism.

SUMMARY OF THE INVENTION

The present invention relates to methods for the generation andselection of Ca²⁺-independent, monospecific antibodies (eitherpolyclonal or monoclonal) specific for defined epitopes covering intact(activation or degradation) cleavage sites of haemostatic proteins suchas, for example, coagulation Factor IX and Protein S, for the isolationof these proteins as intact, uncleaved polypeptides. The strategy ofthis method is based on the notion that disruption of the primarysequence of the target protein leads to a number of changes within theprotein, one being the exposition of the cleavage site itself. Thepresent invention demonstrates that antibodies against epitopes coveringintact cleavage sites may have different affinity for intact and cleavedforms of the epitope, thus allowing separation when applied in achromatographic process. In order to be applicable in purificationprocesses from a variety of sources, the antibodies described in thepresent invention are not only specific for a sequence that provides atarget for limited proteolysis within the coagulation factor to beisolated, but are also Ca²⁺-independent. The method involves theselection of antibodies with appropriate specificity, immobilizationthereof on a solid support, and then contacting a source material withthe immobilized antibody to bind said protein. After washing and elutionwith an appropriate solution, the target protein is recovered in ahighly purified form, in a non-activated, intact state that is ofparticular interest for use in a therapeutic composition for thetreatment of haemostatic disorders.

The process of the present invention is a major breakthrough in thefield of immuno-affinity chromatography in that it achieves uniqueselectivity for intact, non-cleaved target proteins. This allows formethods to obtain intact haemostatic proteins, which can thereafter beformulated into improved therapeutic blood products. These productsprovide safer and more effective agents than are available thus far forthe treatment of patients encountering critical bleeding or clottingepisodes.

Other objects and advantages of the present invention will becomeevident from the following detailed description of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides a method for isolating a haemostatic proteinwhich is susceptible to proteolytic cleavage, from a mixture containingsaid protein, comprising subjecting said mixture to immunoaffinitychromatography using a Ca²⁺-independent antibody which substantiallydistinguishes between intact and cleaved species of said haemostaticprotein.

The term “haemostatic protein” is used herein in a broad sense andcovers not only blood coagulation factors such as Factors IX and VII,but also vitamin K-dependent proteins such as Proteins C and S which arecomponents of a system acting as an antagonist of the coagulationpathway. Further, although this invention covers the isolation ofhaemostatic proteins of any origin, which may be any mammal, it ispreferably applied to the isolation of haemostatic proteins of humanorigin.

The antibody used according to this invention may be a polyclonalantibody, but it is preferred to use a monoclonal antibody. The antibodyis preferably Ca²⁺-independent and directed against an epitope of saidhaemostatic protein which covers an intact proteolytic cleavage site insaid haemostatic protein. The invention also covers the antibody assuch, i.e. a Ca²⁺-independent antibody specific for a haemostaticprotein which is susceptible to proteolytic cleavage, wherein saidantibody substantially distinguishes between intact and cleaved speciesof said haemostatic protein.

In a first set of particularly preferred embodiments of the invention,the antibody is specific for Factor IX and reactive with either anoligopeptide having the amino acid sequence QTSKLTRAETVFPDVD (SEQ IDNO:1) corresponding to the amino acid residues 139-154 of Factor IX, oran oligopeptide having the amino acid sequence QSFNDFTRVVGGEDAK (SEQ IDNO:2) corresponding to the amino acid residues 173-188 of Factor IX, oran oligopeptide having the amino acid sequence ALVLQYLRVPLVDRAT (SEQ IDNO:3) corresponding to the amino acid residues 320-335 of Factor IX, oran oligopeptide having the amino acid sequence TCLRSTKF (SEQ ID NO:4)corresponding to the amino acid residues 335-342 of Factor IX.

According to another particularly preferred embodiment of the invention,the antibody is specific for Factor VII and reactive with anoligopeptide having the amino acid sequence SKPQGRIVGGKV (SEQ ID NO:5)corresponding to the amino acid residues 147-158 of Factor VII.

According to yet another particularly preferred embodiment of theinvention, the antibody is specific for Protein C and reactive with anoligopeptide having the amino acid sequence EDQEDQVDPRLIDGKMTRRG (SEQ IDNO:6) corresponding to the amino acid residues 160-179 of Protein C.

In a further set of particularly preferred embodiments of the invention,the antibody is specific for Protein S and reactive with either anoligopeptide having the amino acid sequence FYPKYLVCLRSFQTGLFTAA (SEQ IDNO:7) corresponding to the amino acid residues 40-59 of Protein S, or anoligopeptide having the amino acid sequence STNAYPDLRSCVNAIPDQ (SEQ IDNO:8) corresponding to the amino acid residues 62-79 of Protein S.

The invention also provides a method for preparing an antibody which isspecific for a haemostatic protein which is susceptible to proteolyticcleavage, wherein said antibody substantially distinguishes betweenintact and cleaved species of said haemostatic protein, comprising thesteps of isolating antibodies which are specific for said haemostaticprotein from animals appropriately immunized to induce said haemostaticprotein-specific antibodies, or from cell cultures producing saidhaemostatic protein-specific antibodies, and screening said antibodiesto select an antibody which substantially distinguishes between intactand cleaved species of said haemostatic protein.

According to the invention, said screening is preferably carried outwith an oligopeptide comprising an amino acid sequence of an epitope ofsaid haemostatic protein which covers an intact proteolytic cleavagesite in said haemostatic protein. Said oligopeptide is preferablyselected from the group consisting of QTSKLTRAETVFPDVD (SEQ ID NO:1);QSFNDFTRVVGGEDAK (SEQ ID NO:2); ALVLQYLRVPLVDRAT (SEQ ID NO:3); TCLRSTKF(SEQ ID NO:4); SKPQGRIVGGKV (SEQ ID NO:5); EDQEDQVDPRLIDGKMTRRG (SEQ IDNO:6); FYPKYLVCLRSFQTGLFTAA (SEQ ID NO:7); and STNAYPDLRSCVNAIPDQ (SEQID NO:8). The invention also covers the oligopeptides as such.

The preparation and characterization of monoclonal antibodies specificfor coagulation factors with intact (activation or degradation) cleavagesite(s) can be realized by the following screening procedure:

1. Definition of a peptide comprising at least part of the cleavagesequence residues −20 to +20, preferably residues −10 to +10, as countedfrom the target peptide bond.

2. Screening of culture supernatants of hybridomas for synthesis ofantibodies binding to the original antigen (present at least partiallyas intact protein) in the absence of Ca²⁺-ions, using standard enzymeimmuno assay, radioimmunoassay, immunoblotting or suitable technology.

3. Rescreening of positive supernatants with defined peptides comprisingrelevant activation and/or degradation cleavage sites. The peptides mayeither be obtained by peptide synthesis, as fragments from the originalantigen, or from other sources including those obtained via recombinantDNA technology. In particular cases the peptides may be coupled tosuitable carrier molecules.

4. Selection and expansion of the appropriate cell line and applicationof the selected antibody in an appropriate affinity chromatographyprocess.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of a haemostatic protein which issusceptible to proteolytic cleavage and a pharmaceutically acceptablecarrier therefor, said haemostatic protein being substantially devoid ofproteolytic cleavage products thereof. More particularly, saidcomposition contains haemostatic protein obtained from a mixturecontaining it by subjecting said mixture to immunoaffinitychromatography using an antibody which substantially distinguishesbetween intact and cleaved species of said haemostatic protein.Preferably, said haemostatic protein is selected from the class ofvitamin K-dependent proteins including Factor IX, Factor VII, Protein Cand Protein S.

The invention also covers a haemostatic protein selected from the classof vitamin K-dependent proteins including Factor IX, Factor VII, ProteinC and Protein S, said protein being substantially devoid of proteolyticcleavage products thereof.

This invention is illustrated in the Examples for Factor IX and ProteinS, but can equally be applied to other haemostatic proteins as will beunderstood by persons skilled in the art.

The Examples provide details of the manner in which the embodiments ofthe present invention may be made and used in order to achieve thegeneration of Ca²⁺-independent monoclonal antibodies substantiallyspecific for intact protein species containing the uncleaved targetsequence. The specificity for the intact species implies that theaffinity for the intact epitope differs substantially from that for thecleaved epitope. This means that the antibody binds intact species witheither higher or lower affinity than the cleaved species, the differenceallowing separation of these species in a chromatographic process. Theseantibodies thus provide novel tools for the isolation of intacthaemostatic proteins. For example, the description given in Example I,while exemplary of the present invention as applied to the generationand selection of monoclonal antibodies against the primary activationcleavage site of Factor IX, and their application for the isolation ofintact Factor IX, is construed to be applicable to other haemostaticproteins, including Protein S, Factor VII and Protein C. Variationswithin the purview of one skilled in this art are to be considered tofall within the scope of the present invention. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare now described.

EXAMPLE 1

Preparation and selection of Ca²⁺-independent monoclonal antibodiesspecific for intact human Factor IX

For the selection of Ca²⁺-independent monoclonal antibodies withsubstantial specificity for intact human Factor IX, peptide No. 1 (seeTable I) comprising the primary activation site Q¹³⁹-D¹⁵⁴ of Factor IXwas synthesized by standard procedures. Immunization, fusion andsubcloning of selected anti-Factor IX positive cultures was performedaccording to established procedures (G. Köhler and C. Milstein, Naturevol 256; 1975, pp 495-497), employing conventionally purified Factor IXas the antigen. Primary screening of the hybridoma cell supernatants wasperformed using a solid phase enzyme-linked immunosorbent assay (ELISA).To this end, purified factor IX was coated to the wells of microtiterplates (Dynatech GmbH, Plockingen, Germany) at a concentration of 0.5μg/ml in 50 mM NaHCO₃ pH 9.5 overnight at 4° C. The plates were washedwith 50 mM Tris-HCl, 154 mM NaCl, 10 mM EDTA, 0.05% Tween-20, pH 8.0 andthen incubated with culture supernatants in the absence of Ca²⁺-ions.Bound antibodies were detected by peroxidase-conjugated goat anti-mouseantibodies using tetramethylbenzidine as substrate. For the secondaryscreening, positive supernatants were rescreened using the same ELISAprocedure, but with peptide No. 1 conjugated to a bovine albumin carrier(5 μg/ml) replacing the Factor IX coating. Two positive cell lines(designated CLB-FIX D4 and CLB-FIX 9) were obtained that were reactivewith both Factor IX and the synthetic peptide No. 1 covering the primaryactivation site. The produced immunoglobulins were purified byion-exchange chromatography and gel filtration using conventionalprocedures.

The monoclonal antibodies CLB-FIX D4 and CLB-FIX 9 were subtyped andidentified as belonging to the IgG1 subclass with a kappa light chainusing a mouse MAb isotyping kit (Holland Biotechnology, Leiden, TheNetherlands). The two monoclonal antibodies were further characterizedwith respect to their affinity for intact Factor IX and the cleavedspecies Factor IXα and Factor IXaβ, employing an ELISA technique asdescribed (Kim et al., J. Immunol. Methods vol 131, 1990, pp 213-222).For both antibodies, the dissociation constant for the complex betweenantibody and intact Factor IX was found to be 1.2 nM, whereas theaffinity for Factor IXaβ appeared to be negligible (dissociationconstant higher than 10 μM). Both antibody CLB-FIX D4 and CLB-FIX 9bound to the cleaved species Factor IXα with substantially higheraffinity (dissociation constants 0.3 and 0.6 nM, respectively) than tothe intact species. These findings demonstrate that cleavage within theepitope of these antibodies results in an alteration of their affinityby at least 2- or 4-fold. The monoclonal antibody CLB-FIX D4 was furtherexamined for its effect on Factor IX clotting activity. A fixed amountof Factor IX (1 μg) was incubated with varying amounts of antibodyCLB-FIX D4 (0-50 μg) at 37° C. for 30 minutes. Subsequently, residualactivity was measured in a one stage coagulation assay (J. J. Veltkampet al., Thromb. Diath. Haemorrh. vol 19; 1968, pp 279-203). In thepresence of a 10-fold molar excess of antibody, Factor IX activity wasinhibited by more than 90%, thus confirming that cleavage within theepitope of antibody CLB-FIX D4 is rate-limiting in Factor IX activation.Immunoadsorbents were prepared by coupling the purified CLB-FIX D4 andCLB-FIX 9 IgG's to CNBr-activated Sepharose (5 mg/ml Sepharose;Pharmacia, Uppsala, Sweden) according to standard procedures. Theimmunosorbents were packed into columns and equilibrated with a bufferconsisting of 20 mM trisodiumcitrate, 154 mM NaCl, 10 mMbenzamidine-HCl, pH 7.4 for use in affinity chromatography as describedbelow.

EXAMPLE 2

Purification of intact Factor IX from prothrombin complex concentrate

The immobilized CLB-FIX D4 IgG was evaluated for use as an immunosorbentfor the isolation of intact factor IX from a prothrombin complexconcentrate prepared by conventional techniques (J. Heystek et al., VoxSang. vol 25; 1973, pp 113-123) as a source material. To 350 ml ofprothrombin complex concentrate, 90 ml were added of a buffer containing0.1 M trisodiumcitrate, 0.77 M NaCl and 0.05 M benzamidine-HCl, pH 7.4.The mixture was applied to a column containing 20 ml of CLB-FIXD4-Sepharose (diameter 2.5 cm, flow rate 25 cm/hr) equilibrated in 20 mMtrisodiumcitrate, 154 mM NaCl, 10 mM benzamidine-HCl, pH 7.4. The columnwas subsequently washed with the same buffer until all unbound proteinwas removed. At this point the buffer was changed to elution buffer (2 MKSCN in equilibration buffer). Fractions were collected and assayed forprotein and Factor IX clotting activity by established procedures (M. M.Bradford, Anal. Biochem. vol 72; 1976, pp 248-254; J. J. Veltkamp etal., Thromb. Diath. Haemorrh. vol 19; 1968, pp 279-203). In the eluate67% of the Factor IX activity was recovered, with a specific activity of356 U/mg. The eluate was free of any detectable Factor IXa activity(i.e. <5 pM) as assayed employing a spectrophotometric method (K.Mertens et al., Thromb. Haemostasis vol 54; 1985, pp 654-660). Inaddition, the non-activated Factor IX product was examined for thepresence of Factor II, Factor X and traces of mouse IgG using standardmethods. The results demonstrate that the residual amounts of thesepotential contaminants in the product are extremely low (see Table II).

Similar results were obtained when the same procedure was performed byusing the immobilized antibody CLB-FIX 9 under identical conditions asdescribed above for antibody CLB-FIX D4. In addition to KSCN, also otherchaotropic salts such as LiCl and NaNO₃ could be used in the elutionbuffer for effectively recovering pure, intact Factor IX.

These results were confirmed by SDS-polyacrylamide gel electrophoresis(K. Weber and M. Osborn, J. Biol. Chem. vol 244; 1969, pp 4406-4412) andWestern-blotting analysis (H. Towbin et al., Proc. Natl. Acad. Sci. USAvol 76; 1979, pp 4350-4355) employing appropriate antibodies againstFactors II, IX, X, Protein C and Protein S. The analysis of flowthroughfractions demonstrated the presence of large amounts of the vitaminK-dependent proteins Factors II, X, Protein C and S. Using polyclonalantibodies against Factor IX, a number of two-chain species representingFactor IX activation products could be visualized as well as someapparently non-bound single-chain species. In contrast, the elutedfractions contained exclusively the non-activated, intact Factor IXspecies. No contaminants could be detected, thereby confirming thequantitative analysis shown in Table II. Affinity chromatographyemploying antibodies CLB-FIX D4 or CLB-FIX 9 thus provides an effectivemethod for the specific isolation of non-activated Factor IX fromprothrombin complex concentrate.

TABLE II Purification of intact Factor IX from prothrombin complexconcentrate (PCC) mouse Volume Protein FIX FII FX IgG Source (ml)(mg/ml) (U/ml) (U/ml) (U/ml) (μg/ml) PCC 350 20.2 39.1 62.5 64.1 n.d.Flow through 440 14.7 10.4 49.7 51.0 n.d. Eluate 73 0.28 98.7 <0.008<0.003 <0.1 n.d.: not determined.

EXAMPLE 3

Selective purification of intact Factor IX from a mixture of partiallycleaved Factor IX species

As affinity chromatography employing antibody CLB-FIX D4 permits thespecific separation of apparently non-activated Factor IX from otherhaemostatic proteins (see Example I), its selectivity for the variousFactor IX cleavage products was evaluated in more detail, with specialreference to Factor IXaβ and its proteolysis-sensitive precursor FactorIXα.

Three mixtures were prepared for subjection to CLB-FIX D4-affinitychromatography, each consisting to a major extent of one specific FactorIX activation product:

(1) Factor IXaβ: Purified Factor IX, as obtained by the method ofExample I, was activated by incubation with purified human Factor XIa.The latter was prepared from Celite-activated human plasma (D. L.Tankersley et al., Thromb. Res. vol 25; 1982, pp 307-317) byimmuno-affinity chromatography using a monoclonal antibody against humanFactor XI (J. C. M. Meijers, Ph.D. dissertation, State University ofUtrecht, 1988, pp 93-108). Factor IX (245 μg/ml) was incubated withFactor XIa (16 μg/ml) in 50 mM Tris, 100 mM NaCl, pH 7.4 containingCaCl₂ (2 mM). After incubation for 2 hours at 37° C., the reaction wasterminated by the addition of EDTA (10 mM final concentration), and themixture was subjected to CLB-FIX D4-chromatography as described below.At this stage, about 90% of the Factor IX had been converted into FactorIXaβ as judged by SDS-polyacrylamide gel electrophoresis andWestern-blotting analysis.

(2) Factor IXα: For the preparation of Factor IXα, Factor IX wasincubated with Factor XIa under the same conditions as described abovefor Factor IXaβ, except that CaCl₂ was replaced by MnCl₂ (6.8 mM) duringincubation. Under these conditions, Factor IXα was found to accumulateas the major cleavage product, while no substantial Factor IXa formationcould be detected. After incubation for 2 hours at 37° C., EDTA wasadded to 10 mM final concentration, and the mixture was subjected toCLB-FIX D4-chromatography. SDS-polyacrylamide gel electrophoresis andWestern-blotting analysis revealed that about 70% of the protein hadbeen converted to the two-chain Factor IXα by a single cleavage at the145-146 position (see Table I).

(3) Factor IX: As a control, the same amount of purified Factor IX wasincubated in the absence of Factor XIa prior to subjection to CLB-FIXD4-chromatography.

These mixtures (2.4 ml) were subsequently applied to an anti-Factor IXCLB-FIX D4 IgG column (volume 3 ml; diameter 1 cm, flow rate 10ml/hour). The column was washed and eluted as described in Example I.The protein content and activity of Factor IX and Factor IXa inflowthrough and eluate were determined using the same methods as inExample I. Table III summarizes these experiments and demonstrates thatFactor IXaβ did not bind to the anti-Factor IX monoclonal antibodycolumn, whereas residual non-activated, intact Factor IX was recoveredin the eluate. The observation that the intermediate cleavage productFactor IXα was not eluted from the affinity column, supports the conceptthat monoclonal antibodies which substantially distinguish betweenintact Factor IX and species that are cleaved at the primary activationsite between residues 145-146, may be employed in a chromatographicprocess for specifically isolating intact Factor IX.

The evidence presented here demonstrates for the first time theutilization of a Ca²⁺-independent monoclonal antibody specific for anepitope comprising an intact cleavage site for the isolation of FactorIX that is, by all the criteria employed herein, essentially free ofother plasma proteins, and at the same time also entirely intact andfree from activated species or activation intermediates. The Factor IXproduct being devoid of any thrombogenic contaminants, it provides amajor improvement over Factor IX preparations heretofore known in theart for use in replacement therapy in patients afflicted withhaemophilia B.

TABLE III Selective purification of intact Factor IX from mixturescontaining cleaved Factor IX species Applied Factor Recovered Factor IXspecies IX Protein (μg) Intact FIX (μg) FIXaβ (μg) species UnboundEluted Unbound Eluted Unbound Eluted Factor 566 62 —⁽¹⁾ 50 550 <0.001IXaβ Factor <10 92⁽³⁾ —⁽²⁾ 85 7 <0.001 IXα Factor 20 580 23 588 <0.001<0.001 IX — intact Factor IX not measurable due to the presence ofFactor IXaβ⁽¹⁾ and Factor IXα⁽²⁾. ⁽³⁾ Number refers to protein eluted in2M KSCN containing buffer; noneluted protein representing Factor IXαcould subsequently be eluted in the same buffer containing 3M KSCN.

EXAMPLE 4

Preparation and selection of monoclonal antibodies specific for intacthuman Protein S

For the selection of Ca²⁺-independent monoclonal antibodies specificallyrecognizing intact human Protein S, peptide No. 7 (see Table I)comprising the primary cleavage site F⁴⁰-A⁵⁹ of Protein S wassynthesized by standard procedures. Immunization, fusion and subcloningof selected anti-Protein S positive cultures were performed employingconventionally purified Protein S (Dahlbäck, Biochem. J. vol 209; 1983,pp 837-846) as the antigen. Primary screening of anti-Protein Smonoclonal antibody secreting cell lines was performed using ananti-Protein S specific ELISA system in the absence of Ca²⁺-ions,essentially as described in Example 1. For the secondary screening,positive supernatants were rescreened using the same ELISA procedure,but with peptide No. 7 replacing the Protein S coating. Two positivecell lines (designated CLB-PS 41 and CLB-PS 52) were obtained that werereactive with both Protein S and the synthetic peptide No. 7 coveringthe cleavage site F⁴⁰-A⁵⁹.

The monoclonal antibodies were further characterized with respect totheir selectivity for intact Protein S within a mixture of cleavedProtein S species. For this purpose, purified Protein S was convertedinto its cleaved form by incubating Protein S (140 μg/ml) with thrombin(0.75 μg/ml) in 50 mM Tris, 150 mM NaCl, pH 7.4. During incubation for 1hour at 37° C., Protein S was cleaved into its two-chain derivative asjudged by SDS-polyacrylamide gel electrophoresis. However, ELISAanalysis (see Example 1) employing the antibodies CLB-PS 41 or CLB-PS 52as the first, immobilized antibody revealed that, in parallel with theextent of proteolysis observed, the recognition of Protein S wasabolished. These findings demonstrate that cleavage within the epitopeof these antibodies results in a substantial alteration of theiraffinity for Protein S. The lack of binding of antibodies CLB-PS 41 andCLB-PS 52 to cleaved Protein S species is in agreement with theirepitope being located at the primary cleavage site F⁴⁰-A⁵⁹ asrepresented by peptide No. 7. The produced immunoglobulins were purifiedand immunoadsorbents were prepared by coupling 5 mg of the purifiedCLB-PS 41 and CLB-PS 52 IgG's to 0.3 g of CNBr-activated Sepharose asdescribed in Example 1. The immunosorbents were packed into columns andequilibrated with a buffer consisting of 20 mM trisodiumcitrate, 154 mMNaCl, 10 mM benzamidine-HCl, pH 7.4 for use in affinity chromatographyas described below.

EXAMPLE 5

Purification of intact Protein S from human plasma

Citrated human plasma (100 ml) was applied to the anti-Protein S CLB-PS52 IgG column (column volume 3 ml; diameter 1 cm; flow rate 10 ml/h).The column was washed with equilibration buffer until all unboundprotein was removed. At this point the buffer was changed todissociation buffer (3 M KSCN in equilibration buffer). Fractions werecollected for analysis of protein content, composition bySDS-polyacrylamide gel electrophoresis and Western-blotting analysis,and of Protein S cofactor activity for activated Protein C (P. C. Compand C. T. Esmon, New Engl. J. Med. vol 311, 1984, pp 1525-1528). Elutionyielded a high-molecular weight species that could be identified as acomponent called C4b-binding protein, which is known to occur in complexwith Protein S in plasma (B. Dahlback and J. Stenflo, Proc. Natl. Acad.Sci. USA vol 78, 1981, pp. 2512-2516). However, no intact Protein Scould be detected in these fractions, indicating that this had remainedbound to the CLB-PS 52 monoclonal antibody column. The buffer waschanged back to the equilibration buffer, and subsequently Protein S waseluted using 6 M Guanidine-HCl in the same buffer. Assays for Protein Sactivity demonstrated that the eluted Protein S product displayed fullbiological activity. Electrophoretic and Western-blotting analysis underreducing and nonreducing conditions revealed that the final product didnot contain any detectable C4b-binding protein or other contaminant, andconsisted entirely of single-chain, intact Protein S.

The evidence provided here demonstrates for the first time that it isfeasible to isolate Protein S in a completely uncleaved, intact state byvirtue of monoclonal antibodies which substantially distinguish betweenintact Protein S and species that are cleaved at the primary thrombincleavage activation site between residues 49-50. When applied in animmunoaffinity chromatography process, a Protein S product is obtainedthat is devoid of any cleaved, inactivated species, and as such providesa major improvement over Protein S preparations heretofore known in theart. This should allow efficient treatment of patients afflicted withthrombosis due to inherited or acquired Protein S deficiency.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims.

16 16 amino acids amino acid unknown unknown peptide NO 1 Gln Thr SerLys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp Val Asp 1 5 10 15 16 aminoacids amino acid unknown unknown peptide NO 2 Gln Ser Phe Asn Asp PheThr Arg Val Val Gly Gly Glu Asp Ala Lys 1 5 10 15 16 amino acids aminoacid unknown unknown peptide NO 3 Ala Leu Val Leu Gln Tyr Leu Arg ValPro Leu Val Asp Arg Ala Thr 1 5 10 15 8 amino acids amino acid unknownunknown peptide NO 4 Thr Cys Leu Arg Ser Thr Lys Phe 1 5 12 amino acidsamino acid unknown unknown peptide NO 5 Ser Lys Pro Gln Gly Arg Ile ValGly Gly Lys Val 1 5 10 20 amino acids amino acid unknown unknown peptideNO 6 Glu Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met 1 510 15 Thr Arg Arg Gly 20 20 amino acids amino acid unknown unknownpeptide NO 7 Phe Tyr Pro Lys Tyr Leu Val Cys Leu Arg Ser Phe Gln Thr GlyLeu 1 5 10 15 Phe Thr Ala Ala 20 18 amino acids amino acid unknownunknown peptide NO 8 Ser Thr Asn Ala Tyr Pro Asp Leu Arg Ser Cys Val AsnAla Ile Pro 1 5 10 15 Asp Gln 40 amino acids amino acid unknown unknownpeptide NO 9 Pro Ala Val Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln ThrSer 1 5 10 15 Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp Val Asp TyrVal Asn 20 25 30 Ser Thr Glu Ala Glu Thr Ile Leu 35 40 40 amino acidsamino acid unknown unknown peptide NO 10 Ala Glu Thr Ile Leu Asp Asn IleThr Gln Ser Thr Gln Ser Phe Asn 1 5 10 15 Asp Phe Thr Arg Val Val GlyGly Glu Asp Ala Lys Pro Gly Gln Phe 20 25 30 Pro Trp Gln Val Val Leu AsnGly 35 40 40 amino acids amino acid unknown unknown peptide NO 11 SerGly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu 1 5 10 15Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser 20 25 30Thr Lys Phe Thr Ile Tyr Asn Asn 35 40 40 amino acids amino acid unknownunknown peptide NO 12 Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro LeuVal Asp Arg Ala 1 5 10 15 Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile TyrAsn Asn Met Phe Cys 20 25 30 Ala Gly Phe His Glu Gly Gly Arg 35 40 40amino acids amino acid unknown unknown peptide NO 13 Tyr Pro Cys Gly LysIle Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys 1 5 10 15 Pro Gln Gly ArgIle Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys 20 25 30 Pro Trp Gln ValLeu Leu Leu Val 35 40 32 amino acids amino acid unknown unknown peptideNO 14 Asp Thr Glu Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly 15 10 15 Lys Met Thr Arg Arg Gly Asp Ser Pro Trp Gln Val Val Leu Leu Asp20 25 30 40 amino acids amino acid unknown unknown peptide NO 15 Val PheGlu Asn Asp Pro Glu Thr Asp Tyr Phe Tyr Pro Lys Tyr Leu 1 5 10 15 ValCys Leu Arg Ser Phe Gln Thr Gly Leu Phe Thr Ala Ala Arg Gln 20 25 30 SerThr Asn Ala Tyr Pro Asp Leu 35 40 40 amino acids amino acid unknownunknown peptide NO 16 Phe Gln Thr Gly Leu Phe Thr Ala Ala Arg Gln SerThr Asn Ala Tyr 1 5 10 15 Pro Asp Leu Arg Ser Cys Val Asn Ala Ile ProAsp Gln Cys Ser Pro 20 25 30 Leu Pro Cys Asn Glu Asp Gly Tyr 35 40

We claim:
 1. A method for isolating haemostatic proteins susceptible tospecific proteolytic cleavage, from a mixture containing said proteinscomprising subjecting said mixture to immunoaffinity chromatographyusing a Ca²⁺-independent antibody which binds intact but does not bindcleaved species of said haemostatic proteins, and isolating thehaemostatic proteins.
 2. A method as claimed in claim 1, wherein saidantibody specifically binds epitopes of said haemostatic proteins whichcovers an intact proteolytic cleavage sites site in said haemostaticproteins.
 3. A method for isolating a haemostatic protein which issusceptible to specific proteolytic cleavage from a mixture containingsaid protein, wherein the method comprises (a) subjecting said mixtureto affinity chromatography using antibodies capable of bindinghaemostatic protein wherein the antibodies are not Ca++-dependent andwherein the antibodies are capable of specifically binding activated andare not capable of specifically binding non-activated species of saidhaemostatic protein, and (b) isolating said haemostatic protein.
 4. Amethod in accordance with claim 3 wherein said haemostatic protein is aVitamin K-dependent protein selected from the group consisting of FactorIX, Factor VII, Protein C and Protein S.
 5. A method for isolating aprotein selected from the group consisting of Factor IX, Factor VII,Protein C and Protein S from a mixture containing said protein whichcomprises subjecting said mixture to immunoaffinity chromatography usingan antibody capable of binding said protein, said antibody being nonCa²⁺-dependent and capable of binding intact but not cleaved species ofsaid protein resulting from specific proteolytic cleavage of the intactform, and isolating the protein.
 6. A method as claimed in claim 3,wherein said antibody is specific for Factor IX and specifically bindsan oligopeptide consisting of the amino acid sequence QTSKLTRAETVFPDVD(SEQ ID NO:1) corresponding to the amino acid residues 139-154 of FactorIX.
 7. A method for isolating an uncleaved haemostatic proteinsusceptible to specific proteolytic cleavage from a mixture containingthe uncleaved haemostatic protein and proteolytic cleavage productsthereof, the method comprising: immobilizing on a solid support a Ca²⁺-independent antibody having substantially higher affinity for theuncleaved haemostatic protein than for the proteolytic cleavageproducts; contacting the mixture containing the uncleaved haemostaticprotein and proteolytic cleavage products with the immobilized antibodyto bind the uncleaved haemostatic protein to the antibody; separatingthe bound uncleaved haemostatic protein from the proteolytic cleavageproducts by affinity chromatography; and isolating the uncleavedhaemostatic protein.
 8. A method according to claim 7 wherein theantibody is specific for an epitope that covers an intact proteolyticcleavage site in the uncleaved haemostatic protein.
 9. A methodaccording to claim 7 wherein the uncleaved haemostatic protein is ablood coagulation factor.
 10. A method according to claim 9 wherein theblood coagulation factor is Factor IX or Factor VII.
 11. A methodaccording to claim 7 wherein the uncleaved haemostatic protein is anantagonist of a blood coagulation factor.
 12. A method according toclaim 11 wherein the antagonist of a blood coagulation factor is ProteinC or Protein S.
 13. A method for isolating an uncleaved haemostaticprotein susceptible to specific proteolytic cleavage from a mixturecontaining the uncleaved haemostatic protein and proteolytic cleavageproducts thereof, the method comprising the steps of: immobilizing on asolid support a Ca ²⁺-independent antibody having substantially loweraffinity for the uncleaved haemostatic protein than for the proteolyticcleavage products; contacting the mixture containing the uncleavedhaemostatic protein and proteolytic cleavage products with theimmobilized antibody to bind the proteolytic cleavage products to theantibody; separating the bound proteolytic cleavage products from theuncleaved haemostatic protein by affinity chromatography; and isolatingthe uncleaved haemostatic protein.
 14. A method according to claim 13wherein the antibody is specific for an epitope that covers an intactproteolytic cleavage site in the uncleaved haemostatic protein.
 15. Amethod according to claim 13 wherein the uncleaved haemostatic proteinis a blood coagulation factor.
 16. A method according to claim 15wherein the blood coagulation factor is Factor IX or Factor VII.
 17. Amethod according to claim 13 wherein the uncleaved haemostatic proteinis an antagonist of a blood coagulation factor.
 18. A method accordingto claim 17 wherein the antagonist of a blood coagulation factor isProtein C or Protein S.